In previous studies examining the role of glucocorticoids and thyroid hormone on the maturation of the Na(+)/H(+) antiporter (NHE3), we found attenuation in the maturational increase in proximal tubule apical Na(+)/H(+) antiporter activity but no change in NHE3 mRNA abundance in either glucocorticoid-deficient or hypothyroid rats. In addition, prevention of the maturational increase in either hormone failed to totally prevent the maturational increase in Na(+)/H(+) antiporter activity. We hypothesized that one hormone played a compensatory role when the other was deficient. The present study examined whether combined deficiency of thyroid and glucocorticoid hormones would completely prevent the maturation of the Na(+)/H(+) antiporter. Adrenalectomy was performed in 9-day-old hypothyroid Sprague-Dawley rats, a time before the normal postnatal maturational increase in these hormones occurs. Nine- and 30-day-old adrenalectomized (ADX), hypothyroid rats had comparable NHE3 mRNA abundance, which was 5- to 10-fold less than 30-day-old ADX, hypothyroid rats that received corticosterone-thyroxine replacement and 30-day-old sham control rats (P < 0.05). Brush-border membrane NHE3 protein abundance was comparable in 9- and 30-day-old ADX, hypothyroid groups and approximately 20-fold lower than both the 30-day replacement and 30-day sham groups (P < 0.05). Similarly, the replacement and sham groups had higher sodium-dependent proton secretion than 9- and 30-day-old ADX, hypothyroid groups (P < 0.05). We conclude that combined deficiency of both hormones totally prevents the maturational increase in NHE3 mRNA and protein abundance and Na(+)/H(+) antiporter activity.